Antibody screening phage display technology

Phage display technology provides an extremely powerful modern way to generate a library of (protein) ligands and, subsequently, screen these ligands for their ability to bind a selected target molecule. The technique, as the name implies, employs filamentous phage (bacteriophage) that replicate in E. coli.

The principle of phage display is presented in Figure 13.2. A library of genes (one of which codes for the protein of interest) is first generated/obtained. These genes are inserted (batch cloned) into a phage library fused to a gene encoding one of the phage coat proteins (pill, pIV or pVIII). The phage are then incubated with E. coli, which facilitates phage replication. Expression of the fusion gene product during replication and the subsequent incorporation of the fusion product into the mature phage coat results in the gene product being 'presented' on the phage surface. The entire phage library can then be screened in order to identify the one(s) coding for the protein of interest. This is usually achieved by affinity selection (biopanning). Biopanning entails passing the library over immobilized target molecules, usually in immobilized column format. Only the phage expressing the protein of desired specificity should be retained in the immobilized column. The bound phage can subsequently be eluted, e.g. by reducing the pH of the elution buffer or inclusion of a competitive ligand (usually free target molecules) in the buffer. Eluted phage can then be repassed over the affinity column in order to isolate those binding the immobilized

Initial gene library 000

Protein 1

Initial gene library 000

Phage replication in E. coli and -^

surface expression of inserted gene product

Phage DNA

Unmodified phages

Phage DNA

Unmodified phages

Protein 2

Phage replication in E. coli and -^

surface expression of inserted gene product

Protein 3

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Supplements For Diabetics

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